IFN-α通过IFI16抑制人脑血管外膜成纤维细胞增殖并促进细胞凋亡

目的:探讨干扰素-α(IFN-α)是否通过诱导干扰素诱导蛋白16(IFI16)表达抑制人脑血管外膜成纤维细胞(HBVAFs)增殖与促进细胞凋亡。方法:应用转染针对IFI16基因的小干扰RNA(siRNA)和/或IFN-α瞬时干预体外培养的HBVAFs。通过蛋白免疫印迹(Western blot)法和Real-time PCR法测定细胞中IFI16、P53、P21蛋白和mRNA表达水平的变化。用MTT法检测细胞活力,流式细胞术测定细胞周期及凋亡。结果:与未干预组比较,IFN-α(2 000~5 000 kU/L)干预HBVAFs后,IFI16蛋白表达水平明显上调,但不同浓度IFN-α组之间无统计学差异。使用2 000 kU/L IFN-α可诱导HBVAFs中P53及P21蛋白和mRNA表达水平上调,同时抑制细胞增殖与促进细胞凋亡。但在转染了IFI16 siRNA的HBVAFs中IFN-α的上述作用受到了抑制。结论:IFN-α抑制HBVAFs增殖与促进其凋亡的作用可能部分与其诱导IFI16表达有关。     

干扰素诱导蛋白p204表达变化对大鼠血管平滑肌细胞增殖及p21表达的影响

目的: 观察干扰素诱导蛋白p204对鼠血管平滑肌细胞(VSMCs)增殖及p21表达的影响,探讨p204在VSMCs增殖调控中的作用及可能机制。方法: 应用干扰素 α(IFN-α)处理和p204基因( Ifi204 )的小干扰RNA(siRNA)瞬时干预体外培养的VSMCs,用MTT法测定细胞活力反映细胞增殖,流式细胞仪分析细胞周期,用半定量RT-PCR 法和Western blotting 分别检测p204和p21的mRNA和蛋白表达。结果: IFN-α可诱导鼠VSMCs p204 mRNA和蛋白表达上调,降低VSMCs细胞活力和细胞周期G₁/S转换,伴p21 mRNA和蛋白表达上调。转染 Ifi204 siRNA可抑制p204和p21表达,提高VSMCs细胞活力和促进细胞周期G₁/S转换。结论: p204表达可抑制鼠VSMCs的增殖,该效应可能与激活p21表达有关。     

干扰素诱导蛋白p204对大鼠血管平滑肌细胞增殖的影响及其机制

目的: 研究干扰素诱导蛋白p204对大鼠血管平滑肌细胞(VSMCs)增殖的影响并探讨其可能的机制。方法: 应用干扰素α(IFN-α)和p204基因(Ifi204)的小干扰RNA(siRNA)瞬时干预体外培养的VSMCs,用MTT法测定细胞活力反映细胞增殖,流式细胞术分析细胞周期,用实时 qRT-PCR法和Western blotting 分别检测mRNA和蛋白表达。结果: IFN-α可诱导大鼠VSMCs p204 mRNA和蛋白表达上调,抑制VSMCs细胞活力和细胞周期G₁/S转换,伴Ras蛋白表达减少,Raf和ERK磷酸化水平下降。转染Ifi204 siRNA可抑制p204表达,提高VSMCs细胞活力和促进细胞周期G₁/S转换,伴Ras蛋白表达增多,Raf和ERK磷酸化水平升高。结论: p204表达可抑制鼠VSMCs的增殖,该效应可能与p204抑制Ras/Raf/MEK/ERK信号通路的激活有关。     

Ifi204基因表达对大鼠血管外膜成纤维细胞凋亡和迁移的影响

目的探讨ifi204基因过表达与沉默对大鼠血管外膜成纤维细胞(VAF)凋亡和迁移的影响。方法原代培养大鼠VAF,利用携带ifi204基因的慢病毒颗粒或空载体慢病毒颗粒感染VAF,分别作为实验组(ifi204 lv组)和阴性对照组(blank lv组)。用ifi204基因小干扰RNA瞬时干预VAF使其沉默(ifi204 siRNA组)、空白小干扰RNA作为阴性对照组(control siRNA组),未经处理的VAF作为空白对照组(negative组)。流式细胞仪检测细胞凋亡,细胞划痕法和Transwell法测定细胞迁移情况,用real-time PCR和Western blot分别检测mRNA和蛋白表达。结果与blank lv组、control siRNA组、ifi204 siRNA组和negative组相比,ifi204 lv组的P204、P53 mRNA和蛋白表达及细胞凋亡率明显上调(P0.05),细胞迁移速度降低(P0.05)。转染ifi204 siRNA可抑制P204、P53的mRNA和蛋白表达(P0.05),提高细胞活力,抑制细胞凋亡(P0.05),加快细胞迁移速度(P0.05)。结论 ifi204基因表达对大鼠VAF细胞凋亡和迁移的影响可能与激活P53表达有关。     

干扰素诱导蛋白16对人脑血管外膜成纤维细胞增殖与迁移的影响及其机制

 目的：研究干扰素诱导蛋白16(IFI16)对人脑血管外膜成纤维细胞（HBVAFs）增殖与迁移的影响及其可能机制。方法：在HBVAFs中转染针对IFI16基因的小分子干扰 RNA (siRNA) 48 h后, 用2×106 U/L 干扰素-α（IFN-α）处理转染IFI16 siRNA细胞24 h。流式细胞术测定细胞周期,细胞划线法与Transwell法测定细胞迁移能力。应用 real-time PCR法和蛋白免疫印迹 (Western blotting) 法分别测定细胞中 IFI16、p53及p21 mRNA和蛋白表达水平的变化。结果：转染IFI16 siRNA后,HBVAFs中IFI16、p53及p21 mRNA和蛋白表达水平下调,增加了细胞G1/S期转换。IFN-α可诱导HBVAFs中IFI16、p53及p21mRNA和蛋白表达水平上调,同时抑制细胞G1/S期转换与细胞迁移,但在转染了IFI16 siRNA的HBVAFs中IFN-α的上述作用受到抑制。结论：IFI16表达可以抑制HBVAFs增殖和迁移,其机制可能与激活p53和p21的表达有关。     

长春新碱降低由转染A20 siRNA促进的弥漫大B细胞淋巴瘤细胞系增殖

目的研究A20小分子干扰RNA片段(siRNA)对弥漫大B细胞淋巴瘤细胞(OCI-LY1)增殖和多药耐药基因(MDR1)表达的影响。方法分对照组、转染组、长春新碱干预组(VCR)组和转染+VCR组。用脂质体转染法将A20 siRNA转入OCI-LY1细胞,MTT法检测细胞对VCR的敏感性;流式细胞计量术检测细胞凋亡;用real-time PCR检测A20及MDR1 mRNA;用Western blot检测细胞内A20、Pgp蛋白及核蛋白NF-κB的表达。结果 1)A20 siRNA转染后,细胞增殖能力增强(P0.05),VCR刺激后转染细胞的增殖曲线下降缓慢。2)转染细胞凋亡率明显低于对照组,VCR刺激24 h后OCI-LY1细胞凋亡率较加药前明显增加(P0.05),VCR组凋亡率比转染+VCR组增高的幅度大(P0.05)。3)转染后,A20 mRNA及蛋白表达明显降低(P0.001),NF-κB蛋白(P0.001)、MDR1mRNA(P0.001)和Pgp(P0.001)表达都明显增高;但MDR1 mRNA和Pgp在药物刺激后表达降低(P0.05),且VCR组较转染+VCR组降低的幅度大(P0.01)。结论 A20 siRNA能有效的增强OCI-LY1细胞内NF-κB的表达,使得MDR1基因及编码蛋白Pgp蛋白增强,从而抑制细胞凋亡、促进细胞增殖;VCR刺激后细胞内MDR1 mRNA和Pgp的表达明显降低,A20 siRNA则减弱了VCR的这一作用。     

针对AKT2的小分子干扰RNA抑制胶质瘤细胞的增殖

目的比较靶向性丝/苏氨酸激酶2(AKT2)不同的小分子干扰RNA构建体对脑胶质瘤细胞增殖的抑制作用.方法选择大鼠AKT2的3个siRNA靶序列构建靶向AKT2的siRNA表达质粒,进行对C6细胞的AKT2表达及增殖抑制的研究,蛋白印迹检测AKT2的表达水平,TUNEL法分析细胞凋亡,流式细胞术分析细胞周期变化,3H-TdR掺入法分析细胞增殖.结果转染siRNA表达质粒组AKT2表达下调72%、88%和91%.各转染组的凋亡率明显增加(x2=34.218,P＜0.001),转染后S期指数较对照细胞明显减少,转染组细胞自第2天起存活率均明显下降(P＜0.01),且各个转染组之间存活率也有极显著性差异(P＜0.01),以尾部调节结构域转染组最显著.结论靶向AKT2的siRNA表达质粒可以特异性地抑制AKT2的表达,显著抑制肿瘤细胞增殖并诱导细胞凋亡.     

Survivin siRNA对人肺腺癌细胞株AGZY增殖及凋亡的影响

目的 探讨靶向survivin基因特异性siRNA对人肺腺癌细胞株AGZY的凋亡、增殖影响以及对其相关机制的探讨.方法 采用RNAi技术靶向干扰AGZY细胞survivin基因的表达,分空白组、转染试剂组、阴性对照组、转染组,用RT-PCR检测各组survivin基因的表达,绘制细胞生长曲线,Hoechst染色检测各组细胞凋亡情况,Western印迹检测各组蛋白survivin、caspase-3、caspase-8、cyclinB1、cyclinD1的变化.结果 Survivin特异性siRNA可在mRNA及蛋白水平有效下调survivin的表达,转染组与其他3组比较差异有统计学意义(F=1785.25,F=38.67,P＜0.05);survivin表达抑制后,与其他3组比较,转染组AGZY细胞数明显减少(F=14999.46,P＜0.05),且转染组细胞出现典型的凋亡改变;与其他3组相比较,survivin特异性siRNA转染组cyclinB1、cyclinD1表达下调(F=109.674,F=313.102,P＜0.05),caspase-3、caspase-8表达上调(F=101.60,F=782.504,P＜0.05).结论 Survivin基因特异性siRNA可以有效下调survivin的表达;survivin表达下降可促进细胞凋亡,抑制细胞增殖;细胞增殖受抑制可能与cyclinB1、cyclinD1表达下降有关,细胞凋亡增加可能与caspase-3、caspase-8表达上调有关.     

转染携带Ifi204基因的慢病毒上调大鼠主动脉成纤维细胞的p204表达

目的观察转染携带Ifi204基因的慢病毒对大鼠血管外膜成纤维细胞p204表达的影响。方法培养大鼠主动脉外膜成纤维细胞(VAF),免疫细胞化学鉴定细胞类型及纯度。用携带Ifi204基因的慢病毒载体系统转染体外培养的大鼠成纤维细胞(Ifi204组),分别以空载慢病毒处理和未处理的大鼠成纤维细胞作为对照组(C组)和空白组(N组)。用q-PCR技术和Western blot检测细胞中p204 mRNA和蛋白表达。结果 p204在N组存在结构表达。与N组和C组比较,Ifi204组的p204 mRNA及蛋白表达均上调(P<0.01)。结论转染携带Ifi204基因的慢病毒可上调大鼠VAF的p204表达。     

NUCB2 siRNA对肝细胞胰岛素信号通路及细胞凋亡的影响

目的探讨靶向NUCB2基因特异性siRNA对大鼠肝细胞IAR20 NUCB2基因沉默效应、对大鼠肝细胞胰岛素信号通路及细胞凋亡的影响。方法构建合成靶向NUCB2基因的siRNA,转染IAR20细胞。Western blot检测PEPCK、G-6-Pase、InsR、IRS-1、Akt的蛋白含量及其磷酸化水平。采用流式细胞术检测细胞凋亡情况,使用RT-PCR检测p53及caspase3 mRNA水平。结果转染siRNA 48 h后,IAR20细胞NUCB2表达明显降低(P均0.05)。PEPCK、G-6-Pase蛋白及mRNA表达量明显增加,同时IR、IRS-1、AKT的磷酸化表达降低(P0.01或P0.05)。IAR20细胞凋亡明显增加,p53及caspase 3 mRNA表达及cleaved caspase 3明显增加(P0.01或P0.05)。结论 NUCB2特异性siRNA能通过下调IR/IRS-1/Akt信号通路加重大鼠肝细胞胰岛素抵抗,并能够通过上调caspase 3及p53表达增加细胞凋亡。     

P300 and sleep-related positive waveforms (P220, P450, and P900) have different determinants

Stimulus factors known to influence the amplitude of the well known endogenous event-related potential (ERP) component P300 were manipulated to determine whether they have the same, or a different, influence on the amplitude of positivities of the sleep ERPs identified as P220, P450, and P900. Behavioral responsiveness and ERPs were recorded as subjects moved from wakefulness to sleep while performing an oddball task. The task consisted of sequential presentation of target and non-target tone stimuli with instructions to respond to targets with a finger--lift response. The probability of the target and non-target stimuli was varied (0.2/0.8, 0.5/.05 and 0.8/0.2) across three test conditions. While subjects were awake, P300 was maximal parietally with amplitude inversely related to the relative probability of the evoking stimulus and directly related to its task relevance. Positive waveforms (P220, P450, P900) recorded in sleep were largest at frontal and central recording sites. P220 and P900 amplitudes were inversely related to stimulus probability. P220 was smaller following target relative to non-target stimuli. Processes underlying P220, P450, and P900 sleep-related waveforms are different from those underlying the P300 component seen in alert wakefulness. The sleep positivities may be state-related waveforms subject to modulation by psychological processes.     

Characterization of SE‐P3, P16, P37, and P47 bacteriophages infecting Salmonella Enteritidis

The aim of this study was to identify and characterize the SE‐P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10–20 min), large burst size (76–356 PFU/cell), and a short burst time (25–35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01–0.0001 and 10⁻⁷, respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20–97.65% in the first 5 min of incubation, and also Ca²⁺ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.     

Functional Differences Between Members of the P300 Complex: P3e and P3b

The main findings of this study bear upon differences in the functional roles of P3b and a shorter latency, more centrally distributed endogenous positive component denoted as P3e. At the present writing, we have observed P3e only in conjunction with P3b. As in the case of P3b, P3e is fully endogenous in that it can be- elicited by omission of a stimulus if stimulus omission conveys relevant information to the subject. It was found that P3e and P3b relate differently to information delivery. Information delivery was manipulated by varying event probabilities and the discriminability of the events. The well known properties of P3b, namely that its amplitude is large when elicited by low probability (high information content) events and is reduced by perceptual difficulty (information loss-equivocation), were replicated in the current study. In contrast to P3b, variation of event probability had no effect upon P3e amplitude, but increased perceptual difficulty markedly reduced P3e amplitude. In addition, two CNV-type negativities were observed in the epochs prior to presentation of the informative signal event: 1) A negativity that was maximal over central scalp related to the subject's prediction that a rare or frequent event would be presented; 2) A negativity that was maximal over occipital scalp related to a stimulus that informed the subject whether the subsequent discrimination of the signal would be easy or difficult. Finally, there was a serendipitous Hading of an apparently new short duration component, tentatively labeled Px, which is elicited by presentation of the signal that informs the subject whether the subsequent discrimination will be easy or difficult.     

Wavelet Analysis of P3a and P3b

Target/standard discrimination difficulty and the degree of stimulus "novelty" were manipulated systematically in a three-stimulus oddball task to assess how these variables affect target and non-target P300 scalp distributions for visual stimuli. Wavelet transformation (WT) analyses were performed on the non-target (P3a) and target (P3b) ERPs to assay how the underlying electroencephalographic (EEG) activity was affected by both the difficulty and novelty factors. When target/standard discrimination was easy, P300 amplitude was higher for the target than the non-target across all electrode sites, and both demonstrated parietal maximums. In contrast, when target/standard discrimination was difficult, non-target amplitude (P3a) was higher and earlier over the frontal/central electrode sites for both levels of novelty, whereas target amplitude (P3b) was greater parietally and occurred later than the non-target components and was generally unaffected by non-target novelty level. The WT analyses indicated that appreciable theta activity was related to the more novel non-target stimuli; primarily target component delta coefficients were affected by the discrimination difficulty variable. The findings suggest that target/standard discrimination difficulty, rather than stimulus novelty, determines P3a generation for visual stimuli but that the underlying theta oscillations are differentially affected by stimulus novelty. WT analysis methods are discussed along with the theoretical and neurophysiological implications of the findings.     

Stimulus context determines P3a and P3b

P300 differences for target (.10), nontarget (.10), and standard tones (.80) were assessed using a three-stimulus oddball paradigm in which participants responded only to the target ( n = 12). Target/standard (easy or difficult) and nontarget/standard (large or small) pitch differences were manipulated orthogonally. In all conditions, target tones elicited a parietal P300, which was affected only by the target/standard discrimination ease. Nontarget in the easy/large and difficult/small conditions elicited a parietal but smaller P300 than the target but in the easy/small condition elicited similar ERPs to the standard. However, nontarget stimuli in the difficult/large condition elicited an anterior maximum and earlier P300 (P3a) component. The findings suggest that target P300s are not influenced by the nontarget stimulus configuration, whereas the nontarget P300 outcomes are determined directly by the stimulus context. The theoretical implications are discussed.     

The formation of P particle increased immunogenicity of norovirus P protein

As a commentary on a recently published paper in Immunology, this article summaries the principle of norovirus P particle as a promising vaccine against noroviruses. It emphasizes the importance of P particle formation in the immune enhancement of the vaccine and methods for production/verification of high quality P particles which may be easily neglected by researchers.     

The Development of Visual P3a and P3b

The relationship of visual P3a and P3b to age and neuropsychological performance was investigated in 26 healthy children (6.8–15.8 years) and 129 adult volunteers (20.0–88.8 years). Within the sample of children, an effect of age on midline topography was observed, with higher frontal amplitudes in the youngest compared to the oldest children. Increasing age was associated with lower P3a and P3b amplitude and shorter P3b latency at Fz. Performance on neuropsychological tests (matrix reasoning from WASI, digit span from WAIS, word order and hand movement from Kaufman) was only weakly associated with measures of P3a and P3b. The analyses were then repeated with the full life-span sample (n = 155). It was found that for P3a, amplitude decreased and latency increased with age. For P3b, the pattern was more complex, with a nonlinear amplitude reduction and no latency change with age. It appears that the development of P3a in children represents the start of processes that later continue in the adult life-span, but that the automatic processes indexed by P3a seems to mature earlier than the controlled processes reflected by P3b. Finally, it was demonstrated that the relationships between neuropsychological test scores (matrix reasoning, digit span) and P3 parameters were complex, following a mix of linear and nonlinear patterns. It is suggested that the neuropsychological significance of the different P3a and P3b parameters may change from childhood to the adult life-span.     

Mutual derepression in the P2–P4 bacteriophage system

P4 lysogens are derepressed by infection with P2 as is evident from the induction of P4 phage production by P2 infection of P4 lysogens. Mutations interfering with P2 DNA synthesis greatly increase the P4 yield, but DNA replication by the infecting phage does not preclude P4 induction. Of four P2 mutations known to block spontaneous phage production by P2 lysogens, three (cox-2, cox-3, cox-4) also prevent derepression of P4 lysogens by P2. These studies also revealed that, under certain conditions, P4 can complement P2 mutants deficient in gene B function which is required for P2 DNA replication. P2 lysogens are derepressed by infection with P4. This derepression leads to a P2 gene A-dependent P2 prophage replication and, in P2 lysogens mixedly infected with P2 and P4, also to the replication of the DNA of the coinfecting P2. If the derepressing P4 is deficient in gene a function, then derepression of the P2 lysogen will lead to the production of 10 or more P2 phages per cell, provided that P2 prophage excision is enhanced by the P2 nip mutation or is bypassed by infecting the P2 lysogen with P2 as well as with P4. Gene α proficient P4 interferes with the P2 phage production by P2 lysogens; possible causes for this interference are considered, including P4 DNA replication which is known to require gene a function.     

Substance P and substance P receptors in bone and gingival tissues

Substance P (SP) is an important member of the tachykinin family of neuropeptides, which work as neurotransmitters or neuromodulators. Recent advances in the analysis of SP receptors, particularly the neurokinin-1 receptors (NK1-Rs) that have high affinity for SP, have demonstrated that they are distributed not only in the cells of the neuronal or immune systems but also in peripheral cells. Therefore, the effect of SP and its cellular receptors is not limited to the nervous or immune systems, but is more extensive than previously appreciated. SP-like immunoreactive (SP-LI) axons have been localized in both bone and gingival tissue, and SP receptors are widely distributed in osteoclasts, osteoblasts, and junctional epithelial cells, as well as in neutrophils and endothelial cells. The distribution of SP-LI axons and SP receptors suggests that SP may directly modulate bone metabolism and gingival tissue functions through SP receptors.